Download Background and Overview of Comparative Genomics

Background and Overview of Comparative Genomics
Jennifer A. Marshall Graves
Comparative genomics—the cross-referencing of information on genome organization between species—provides an
additional dimension to the Human Genome Project and can
derive much information from it for the benefit of animal
health and animal breeding. Arrangements of genes and other
DNA sequences may be determined by a variety of genetic
and physical techniques, at resolutions from the gross cytological level to the level of the single base pair. Gross arrangements and rearrangements can also be charted by comparative chromosome painting. Genome organization may
then be compared across mammal—and other vertebrate—
species. Genetic mapping is well advanced in several livestock species as well as rodent model species, and outline
maps are available for at least 30 mammal species in 8 orders. At the time of this writing, maps are being rapidly
constructed for chicken and fish species. Comparisons, even
over vast evolutionary time scales, show that the mammal
genome—indeed, the vertebrate genome—has been highly
conserved. Thus information about location and function of
genes is directly transferable across species and should
greatly speed up the search for genes that specify inherited
diseases in domestic mammals and humans as well as genes
that specify economically important traits.
INTRODUCTION
The map and base sequence of the human genome will stand
as a monument to 20th century technical innovation and doggedness. A detailed gene map is already available, physical
maps encompass several chromosome regions and 2 whole
chromosomes, and sequencing is accelerating toward a
scheduled completion date of 2003, 2 yr earlier than originally targeted. At the time of this writing, the human map
is already being used for locating genes that act to bring
about variation in outward characteristics and disease states
(phenotypes).
The prodigious output of the Human Genome Project can
be directly applied to increase our knowledge of, and control
over, every mammalian species of use and interest to humans
and can even be applied to other vertebrates such as birds and
fishes. Comparative genomics can also use knowledge gained
from other animal species to advance the goals of the Human
Jennifer A. Marshall Graves, Ph.D., is Professor and Head of the School of
Genetics and Human Variation, LaTrobe University, Melbourne, Australia.
48
Genome Project and to contribute to an even broader goal—
understanding the evolution of the human species.
GENES AND GENOMES
The genome is defined as the full set of genes that defines an
organism. Mammals and other vertebrates, being diploid,
have 2 full genomes in each cell, 1 from the sperm and 1
from the egg.
Genome Function and Organization
Oddly, considering their unsuitablility as experimental organisms, humans are our mammal type-species. The human
genome is by far the best known genome of any mammal and
so is the obvious point of all comparisons. It contains approximately 70,000 genes, of which about 10% have been
identified and mapped and another 20% have been ordered
as short (100-base pair) runs of identifiable unique sequence.
These genes have functions in basic metabolism or in determining specialized characteristics of the thousands of different cell types that make up the human body. The protein
product of a gene may act as an enzyme (or an enzyme
subunit), a transport molecule, a structural protein, a hormone, or a regulator in any of the myriad biochemical reactions of the body.
Each gene consists of a sequence of DNA, which is transcribed into RNA in the appropriate tissue or organ, then
translated into a single polypeptide in the cytoplasm of cells.
In this process, the sequence of nucleotides (bases) along the
double helical DNA molecule is translated through the fabled
genetic code in which each amino acid in the polypeptide
product is specified by a triplet of bases. The average length
of coding portion of genes is about 1000 base pairs (1000
base pairs = 1 kilobase, or kb). Between the genes are long
stretches of DNA often full of repetitive sequences, which
have no obvious function and may simply represent genetic
junk. Even within genes, intervening sequences of noncoding
DNA ("introns") are interspersed between coding regions
("exons"), and are spliced out of messenger RNA after transcription. Overall, only about 3% of the mammalian genome
is coding DNA.
In all organisms, genes are parts of immensely long DNA
molecules. Thus genes are arrayed linearly along chromosomes, and this linear array can be represented as a linear map.
ILAR Journal
The entire mammalian genome, which represents more
than 1 m of DNA, is divided up into smaller lengths for ease
of handling during cell division, when the genome is replicated and the daughter DNA molecules are segregated. These
DNA molecules constitute the chromosomes that we can see
as staining bodies when they are coiled and packaged up
with protein, ready to be apportioned at mitosis.
Chromosomes may be photographed or imaged at mitosis, then arranged as a karyotype in order of descending size.
Because vertebrates are diploid, there are 2 copies of each
chromosome. Chromosomes are distinguished, as well as by
their size, by the position of the centromere, which remains
undivided, attaches spindle fibers at mitosis, and appears as a
constriction. Chromosomes may also be linearly differentiated by several methods (such as Giemsa or G-banding1) that
induce specific patterns of dark and light bands along their
length. This pattern reflects the underlying sequence organization, and G-band patterns are therefore diagnostic for each
chromosome and consistent within the species.
Genome Comparisons
In all mammals, the genome is basically the same size, comprising approximately 3300 million base pairs (or Megabases
[Mb]) and about the same number of genes (approximately
70,000). These genes represent much the same set as in humans, performing the same housekeeping and specialized
tasks. As for the human genome, other mammalian genomes
are composed largely of noncoding, mostly repetitive sequences. Compared with those of mammals, the genomes of
birds are only about 1/3 the size, and the genome of the
pufferfish (Fugu) is especially compact at about 1/8 the mammal genome size, although it appears to share a similar set of
genes.
The equivalent genome is divided differently in different
mammal species. Haploid chromosome numbers (that is, the
number in a single genome) range from n=3 large chromosomes in the Indian muntjac to n=67 small chromosomes in
the black rhinoceros. This karyotypic variation—the different numbers, sizes, and shapes of chromosomes in different
species and the difficulties of comparing G-band patterns—
for a long time led to a belief that many rearrangements had
scrambled gene orders beyond recognition in different mammal lineages. However, comparative mapping over the last 2
decades has shown that the mammalian genome is much
more conserved than was apparent from cytogenetic comparisons, and this conclusion is now being reinforced by
direct observations of comparative chromosome painting.
The remarkable conservation of the mammal genome—in-
1
Abbreviations used in this paper: BAC, bacterial artificial chromosome;
FISH, fluorescence in situ hybridization; G-banding, Giemsa banding;
MYA, million years ago; PCR, polymerase chain reaction; QTL, quantitative trait locus; RFLP, restriction fragment length polymorphism; YAC,
yeast artificial chromosome.
Volume 39, Numbers 2 and 3
1998
deed, the vertebrate genome—has resulted in the ready transfer of information between different mammal species and the
belief that deducing the shape of an ancestral mammalian
genome—maybe even an ancestral vertebrate genome—is
not a vain hope.
Comparative genomics includes comparisons of every
type of gene map at every level from the cytological to the
molecular. These methods are described and explained below in Markers for Genome Analysis. The text summarizes
the value of such comparisons in practical outputs for animal
health and animal breeding as well as for using animals as
models for human disease. The summary also describes our
first attempts to solve the great evolutionary jigsaw puzzle—
to deduce the ancestral arrangements from which the genomes of all living mammals derived.
MARKERS FOR GENOME ANALYSIS
Different strategies may be used to detect genes and DNA
sequences and to compare their positions and arrangement in
different species. Classically, genes were recognized by their
effects on the phenotype, and a genetic map was constructed
by observing the patterns of segregation among the offspring
of parents differing in 2 or more characteristics. Genetic maps
still rely on inherited differences, but these may be recognized at the molecular as well as the phenotypic level. Physical maps describe the physical location of a gene or a DNA
sequence on a chromosome or chromosome region, with
greater or lesser resolution. Maps on a molecular scale can be
constructed with the base sequence of the region at their
endpont.
Different types of maps require different types of gene
marker. Genetic (linkage) mapping relies on intraspecies
variation (alleles) at a particular gene locus, whereas somatic
cell genetic mapping requires interspecific variation, and
physical mapping techniques require a cloned DNA probe.
The availability of molecular and phenotypic differences between alleles has vastly increased the ease of genetic mapping, especially in humans, where information is compiled
from limited and uncontrolled crosses. Importantly, this
availability has also made it easy to line up genetic and physical maps of the same species and to move between them in
physically isolating a target gene.
Molecular Markers
Molecular markers have been developed as the result of gene
cloning—from the ability to isolate and replicate small pieces
of large genomes by splicing them into small self-replicating
DNA molecules of bacteria or virus vectors. Vectors have
different characteristics that make them suitable for different
types of study. At the lower end are lambda virus vectors,
which accept 15 to 20 kb of insert DNA, and bacterial plasmids and cosmids, which can accommodate slightly more.
At the upper end are bacterial and yeast artificial chromo49
somes (BACs1 and YACs1), constructed by stringing together
the components of normal chromosomes, which may accept
pieces of foreign DNA up to a few megabases. The entire
genome of a mammal can be fragmented into appropriate
sized pieces, which are inserted randomly into vectors and
propagated as mixtures (libraries) of colonies or plaques on a
Petri dish. The vector carrying a particular sequence can then
be isolated by cloning the progeny of a single virus, bacterium, or yeast.
One variation is to start with messenger RNA prepared
from a particular tissue, make DNA copies (cDNA) and splice
them into a vector. These cDNA libraries provide intronless
copies of all the genes active in a particular tissue, so they
enable us to concentrate on the minority of the genome that
codes for protein. Very rapid progress has been made in identifying all the expressed genes in the human genome as minimally sequenced expressed sequence tags ("ESTs") whose
unique position can be determined on the map.
An alternative for isolating a single sequence from a complex genome is to replicate it selectively, exploiting the predilections of the DNA replication system in the polymerase
chain reaction (PCR1). Under in vitro conditions, DNA polymerase will replicate DNA only when provided with 2 short
(approximately 24 bases) "primer" sequences, which are constructed to be complementary to short sequences on either
side of the target. Over a number of replication cycles, PCR
will therefore amplify a target sequence between 2 primers
in a chain reaction.
Variation in DNA markers can be detected in a number
of ways. Base differences can be identified as restriction
fragment length polymorphisms (RFLPs1) if they happen to
fall within a recognition site for a restriction enzyme. Otherwise, they may be detected by hybridizing with allele-specific oligonucleotides, or allele-specific primers for PCR,
which bind only if there is a perfect match. Single base differences in a DNA molecule can also be detected by single
strand conformation polymorphism.
Especially variable and therefore exceedingly useful
markers have been repetitive sequences, which frequently
differ in the number of tandem repeats. Initially, variable
numbers of tandem repeats (or minisatellites) were detected
by hybridizing with the (often lengthy) core sequence and
detecting different sized bands on a Southern blot. A very
sensitive detection system is now available to detect variation in the numbers of very simple sequence repeats (di-,
tri-, or tetranucleotides) by PCR from primers lying outside
the repeat stretch. The product length is assessed on an
acrylamide gel that can separate alleles differing in only 1 or
a few repeats. These simple sequence length polymorphisms,
commonly known as microsatellites, have revolutionized
genetic analysis of complex genomes.
Type I and Type II Markers
Many of the markers most useful for constructing a detailed
linkage map are anonymous DNA sequences, most of which
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are derived from the majority noncoding and therefore more
variable class of DNA (type I markers). However, these
highly polymorphic markers are of very limited use for comparisons between genomes because their variability makes it
impossible to detect homology across species. Although they
may be employed in linkage mapping in closely related species (for example, cattle microsatellites have been valuable
for constructing a sheep linkage map; Broad and others
1998), they are not likely to recognize homologous sequences
between more distantly related species. The most useful
markers for comparative genetics are the highly conserved
coding genes (type II markers), which may show greater than
90% sequence homology within the exons between all mammals, even all vertebrates, and be recognizable even in
Drosophila and yeast.
Markers for mapping coding genes must be found in
variation of the properties of a gene, its protein product, or
the resulting phenotype. Changes in the gene product are
usually detected electrophoretically as altered charge on the
protein. Changes in the base sequence of the gene itself can
be detected as RFLPs or simple sequence length polymorphisms (microsatellites) within the gene. The problem with
type II markers is that the very conservation of coding genes
means that there are few polymorphisms available for placement on the genetic map. A solution is to screen introns for
sequence differences or microsatellites that can be recognized by amplifying from conserved exon sequences. Sets of
conserved "universal" primers (Venta and others 1996), or
comparative anchor-tagged ("CAT") sequences (Lyons and
others 1997), made by designing primers to exon sequences
conserved between at least 2 species, have been of considerable use for comparative mapping, at least in some mammalian species, although many "universal" primers have proved
to have limited applicability.
A particularly useful strategy to increase the supply of
polymorphic markers has been to use hybrids between
closely related species. This has been spectacularly successful in constructing a detailed linkage map of the mouse genome using Mus musculus x Mus spretus crosses (Davisson
and others 1998), but the same idea lies behind the development of crosses between bovine species, deer species, cat
species, and subspecies of the tammar wallaby. The same
strategy was used 20 yr ago to map some fish species
(Morizot and others 1998).
Criteria of Homology and Nomenclature
Problems
Genes often belong to large gene families with similar sequences. A big problem for comparative mapping is distinguishing between orthologues (homologous genes in different species, such as alpha globin in human and mouse) and
paralogues (genes within the same species descended from
the same ancestral gene by duplication and divergence, such
as human alpha and beta globin). It has therefore been necessary to develop specific criteria by which orthologues may
ILAR Journal
be recognized in different species at the DNA, protein, or
phenotype level (CGOW 1996). The most stringent of these
criteria are
•
•
•
at the nucleotide level, similar nucleotide sequence or at
least cross-hybridization to the same DNA probe. Similar transcription profile is helpful supplementary data but
cannot be regarded as diagnostic because there are too
many exceptions.
at the protein level, similar amino acid sequence. Similar
biochemical properties, expression profile, and immunological cross-reaction are regarded as helpful but less
robust.
at the phenotype level, complementation of function is a
much superior criterion than similarity of mutant phenotype.
It is a sign of the recognition of the extent of genome
conservation that an important criterion of homology has
now become conserved map position, which can often be
used to distinguish between paralogues. Authors must always state the criteria by which homologues have been
recognized.
Gene nomenclature has been a continuing problem in
comparative gene mapping, although this has been mitigated
by agreements by most species groups (even rat) to adopt
human nomenclature where homology is clear. Mouse nomenclature still retains symbols originally descriptive of
mutant phenotypes. Although this practice is too well established to make changing practicable, the same symbol is now
used where possible for new genes described in human and
mouse.
Species designation also remains very inconsistent in
comparative genomics publications. The convention recommended by the Comparative Genome Organization Workshop is a 4-letter code derived from the species name and
italicized to reflect standard species nomenclature. Examples
include Hsap for Homo sapiens, Mmus for M. musculus,
Meug for the tammar wallaby (Macropus eugenii), and Ggal
for the chicken (Gallus gallus).
Terms particularly useful for comparative gene mapping,
as defined by the Comparative Genome Organization Workshop (1996) are
•
•
•
•
homology—regions containing arrays of homologous
genes in different species;
conserved synteny—syntenic associations of the same
genes in different species, regardless of gene order or
interposition of other genes;
conserved segment—syntenic associations of contiguous genes in different species. The shortest conserved
segment in different species has been called a SCEUS,
for "smallest conserved evolutionary unit segment";
and
conserved order—3 or more homologous genes in the
same order in different species.
Volume 39, Numbers 2 and 3
1998
MAPPING METHODS
Two types of map describe a genome: genetic maps, which
are derived from recombination frequencies between genetic
markers; and physical maps, which are constructed from information about the physical location of genes on chromosomes. The 2 maps may be aligned if they share markers.
Genetic Maps
Genetic maps were devised in the early 1900s by Thomas
Hunt Morgan and his students, using crosses in fruit flies.
This discovery followed observations in the early 1900s that
some genes acted as if they were "linked" together in the
offspring, whereas others segregated independently, according to Mendel's laws. Linkage was explained by the tendency of 2 markers on the same parental chromosome to be
passed on together, in contrast to markers on different chromosomes, or far apart on the same chromosome, behaving
independently at meiosis. Linkage reflects the physical reality of crossing over between 2 homologous chromosomes at
meiosis. Morgan observed that the amount of recombination
between 2 gene loci varied widely, and his student guessed
that the recombination percentage between 2 loci was related
to how far apart they were physically.
The technique for genetic mapping is therefore to mate
parents differing in 2 or more traits (that is, having different
alleles at 2 or more loci) and then to score the patterns of
segregation of parental alleles among the offspring. 2 genes
on different chromosomes will randomly segregate at meiosis, so that 1/4 of the offspring will show each of 4 combinations of alleles at the 2 loci. However, if genes are close
together on the same chromosome, parental combinations of
alleles will pass to the offspring together, unless they are
separated by recombination. Since the probability of recombination increases as the physical distance between genes,
the recombinant percentage progeny provides a measure of
the genes' relative distance apart. The unit of measurement is
called a centiMorgan (cM) after Morgan. For markers close
to each other (where the complication of double recombination can be ignored) 1 cM = 1 % recombination.
If the parents differ at 3 or more loci, recombination
between them is additive, allowing for multiple recombination. This allows the 3 loci to be placed in a linear array in
which recombination percentages represent the relative distances between the loci in this "linkage group." A map can
be built up in stages from different crosses in different laboratories, and ultimately, the linkage group describes the gene
order along a whole chromosome.
Linkage maps depend utterly on the availability of polymorphisms—the existence of 2 or more alleles at a locus.
This was traditionally the great limitation of mapping, but
the availability of highly polymorphic DNA markers (RFLPs
and especially microsatellites) has revolutionized genetic
mapping in many animal species.
51
Physical Maps
Genes may be assigned to physical positions within chromosomes or chromosome regions by somatic cell genetics, radiation hybrid mapping, and in situ hybridization. A molecular description of the genome, given by restriction mapping
and nucleotide sequencing, also qualifies as physical mapping, but is considered below separately.
Somatic cell genetics was developed in the 1970s and in
situ hybridization in the 1980s. Neither method requires a
breeding colony, or even a live animal, as long as cell samples
are available. Nor do they require polymorphic markers,
since somatic cell genetics uses interspecific variation and in
situ hybridization requires only a cloned sequence big enough
to produce a signal. However, both methods represent low
resolution techniques. Somatic cell genetics simply establishes synteny groups and assigns them to a chromosome, but
does not specify position or order; and in situ hybridization
provides a rough cytological localization on a chromosome.
Somatic cell genetic analysis uses viable hybrid cells
derived from fusion of somatic cells from different species.
It depends on the observation that chromosomes are lost from
only 1 of the 2 parental sets (at the time of this writing, we
still do not know the reason). For example, rodent-human
hybrids segregate human chromosomes, so that it is possible
to derive a hybrid panel uniquely representing each human
chromosome. Hybrids all retain and express the full set of
rodent genes; however, a hybrid will retain and express only
the human genes on the particular human chromosomes retained in that hybrid. Thus, by detecting patterns of presence
and absence of human markers in a set of hybrids and correlating these with the patterns of the presence or absence of
particular chromosomes, it is possible to assign a human
gene to a particular human chromosome. Some regional mapping is possible using hybrids that retain only portions of a
chromosome.
The method described above was developed particularly
for humans but has been used extensively since the early
1980s to assign genes to chromosomes in cattle, cat, and
marsupial species. Hybrid panels have also been developed
for more exotic species such as mink, arctic fox, and vole
(Rubstsov 1998; Serov 1998) and most recently for shrew
(Nesterova and others 1998).
Radiation hybrid mapping is an extension of regional
mapping. Hybrids are constructed from donor cells (cells
from the species to be mapped or cell hybrids bearing a
single chromosome of the species to be mapped) that have
been lethally irradiated to cause chromosome fragmentation.
Hybrids therefore contain only small regions of the irradiated donor genome incorporated into chromosomes of the
unirradiated parent. These radiation hybrids are more likely
to bear 2 genes if they are physically close together on a
chromosome, so the frequency of concordance of markers
may be used as a measure of their physical proximity.
In situ hybridization is a method by which a cloned probe
labeled with radioactive isotope or fluorescent tag is bound
specifically to the DNA sequence to which it is complemen52
tary, within the framework of the chromosome fixed to a
microscope slide. Radioactive signal is detected by autoradiography. Fluorescent tag is bound indirectly to the probe
by layers of specific antibodies that detect molecules bound
to the DNA (for example, biotinylated probe is bound to
avidin and detected by fluorescent antibodies). Fluorescence
is detected by a sensitive ultraviolet light microscope. Fluorescence in situ hybridization (FISH1) is remarkably sensitive
as long as the probe is homologous and long and the background of repetitive sequence is suppressed by competing
with unlabeled whole DNA or the repetitive fraction. It provides a localization to a region about 1% of the genome
length (that is, approximately 30 Mb). Different fluorescent
dyes produce signal at different wavelengths so that if the
efficiency is high enough, 2 or more colors may be used to
identify different sequences within the same cell.
Chromosome Painting
Chromosome painting is a fluorescence in situ hybridization
technique that differs from FISH in that it uses a unique
DNA probe derived from a whole chromosome or chromosome region. Chromosomes from a species may be physically separated by flow sorting or microdissection, as described by Ferguson-Smith and others (1998). DNA from a
single chromosome may then be PCR amplified using degenerate oligonucleotide primers so that all sequences are
represented. When a single chromosome paint is applied to
chromosome preparations from the same species under suppression hybridization conditions (so that repetitive sequences shared between many chromosomes are not detected), only the 2 copies of that chromosome are hybridized.
The regions that hybridize to the paint are then detected by a
fluorescent tag (in the same way as for FISH) and appear as
a colored region.
Paints have been prepared from each of the flow-sorted
human chromosomes. In the same way, single chromosome
paints have been prepared from all the chromosomes of the
cat, mouse and several farm mammals as well as marsupials
and chicken (Ferguson-Smith and others 1998). Paints can
also be prepared from regions of chromosomes—even single
G-bands—by microdissection.
A single chromosome paint from 1 species may then be
applied to chromosome preparations of another species under suppression hybridization conditions so that it binds only
to homologous regions. A pattern of regions homologous
between species may be obtained. Again, different dyes may
be used to produce signal at different wavelengths. Different
combinations of 3 dyes can produce 24 distinguishable signals so that painting with all the human chromosomes simultaneously may be performed (Wienberg and Stanyon 1998).
Comparative chromosome painting (or ZOO-FISH) has
been most effective when performed between species reasonably closely related such as human and apes (Wienberg
and Stanyon 1998), mouse and rat (Ferguson-Smith and others 1998), or 2 species of kangaroo (Toder and others 1998).
ILAR Journal
Good signal has also been produced by hybridizing human
paints onto carnivore, ungulate, and even insectivore chromosomes (summarized in Glas and others 1998). Painting
rodents with human paints has been more of a challenge
because there have been many more rearrangements as well
as more sequence divergence.
Painting is an extremely direct way to assess the amounts
of rearrangement between 2 species, with the advantage over
comparative mapping of providing direct information on homologies over an entire genome in about 1 wk. Such information would otherwise require a relatively detailed comparative map, taking many years to construct. Chromosome
painting can rapidly extend results from comparative mapping. For instance, the cat map covers 50 to 60% of the
genome and the painting, more like 90%.
However, the resolution of the method is a limitation.
Painting between relatively closely related species can detect
rearrangements as small as 5 to 10 Mb (Wienberg and
Stanyon 1998). Resolution can be improved by using reciprocal painting between 2 species to ensure that small unpainted regions, which are difficult to detect in a brightly
fluorescing background, are not overlooked. The inability to
detect rearrangements within conserved chromosome blocks
is also a limitation.
Mapping at the Molecular Level
Physical mapping using somatic cell genetic or in situ hybridization techniques has limited resolution. At the other
end of the scale are methods that provide physical information at a molecular level: restriction mapping of cloned sequences, contig construction and, ultimately, complete base
sequencing.
Restriction mapping of a region of DNA is based on the
recognition of specific base sequences (usually 4- or 6-base
palindromes) by restriction enzymes, liberating restriction
fragments of specific sizes that can be visualized after separation by gel electrophoresis. Different enzymes cleave the
same DNA into overlapping fragments. The arrangement of
the restriction sites can be deduced by comparing the fragments released by digestion with restriction enzymes singly
and in combination. Long range restriction maps can also be
produced using enzymes that cut infrequently at 8-base, 10base, or even longer recognition sites. In this way, cloned
pieces of DNA (small lambda clones up to megabase-sized
YACs) may be mapped and compared. Very detailed patterns of cut sites can be recognized in overlapping clones, so
a map of contiguous regions of DNA (contigs) can be constructed.
The ultimate information about a region of the genome is
its nucleotide sequence. Sequencing of fragments (usually
subloned restriction fragments or target sequences between
PCR primers) of a genome is relatively straightforward, although it is still a massive undertaking for more than just a
gene and its surroundings. The ultimate goal of the Human
Genome Project—to sequence the entire genome by 2003—
Volume 39, Numbers 2 and 3
1998
appears realistic in view of accelerating progress, with large
tracts of several human chromosomes sequenced.
However, for other mammalian genomes, sequencing is
a rather inefficient way of obtaining information about coding regions, since a majority of the genome is noncoding,
and there is only 1 gene on the average every 40 to 50 kb.
Thus, mass sequencing is unlikely to be undertaken for any
other mammal except mouse, in which a sequence-ready
physical map is already being constructed at the time of this
writing (Davisson and others 1998). Sequence comparisons
in mammals are therefore usually made on a gene-by-gene
basis.
Mass sequencing of random clones is practicable for the
compact genome of the pufferfish Fugu, which has a gene
density of 1 per 6 kb. Since breeding this species is difficult,
sequence scanning is the method of choice, and direct scanning of (usually human or mouse) databases is the method of
comparison with mammals (Elgar and Clark 1998).
Putting the Maps Together
The potential for cross-referencing the different types of
maps is important because it allows us to move from a genetic map (which may contain markers known only as phenotypes, like many genetic diseases) to a physical map and
ultimately to a molecular map. This cross-referencing provides the tools to identify and physically isolate the gene.
What limits this comparison is the type of marker from which
the map is built up; genetic maps are composed largely of
highly polymorphic DNA markers (type I), whereas physical
maps are composed largely of cloned genes (type II). It is
therefore wise to include coding genes within genetic maps
so that they can be transferred to physical maps and compared across species.
A physical description of the DNA molecule that constitutes a chromosome—or an entire genome—can be described
at several levels of resolution. At the cytological level, some
detail (to approximately 10 Mb) is given by medium-resolution G-banding and chromosome painting. A low level of
resolution of gene location is provided by somatic cell genetics and in situ hybridization (approximately 30 Mb), but linkage mapping can resolve markers as close as 1 cM, which is
equivalent to 1 to 2 Mb. The molecular level represents the
other extreme level of resolution. Restriction maps provide a
linear array of restriction enzyme cut sites, usually several
per kilobase over distances of tens of kilobases. The ultimate
in resolution is the base sequence.
A gap exists between the resolution offered by genetic
and physical mapping and molecular characterization. This
gap can now be filled by large insert clones (BACs and
YACs), and contigs built up by their overlap that span up to
whole chromosomes. Radiation hybrids also provide an intermediate level of resolution where, at least in human, radiation hybrid mapping has permitted the physical ordering
of several thousand human loci. This method offers the potential to order large or small chromosome segments isolated
53
in a somatic cell hybrid, potentially providing the framework
for building a YAC contig.
DEPTH AND BREADTH OF INFORMATION
All these methods have been used to various extents, depending on the resources available, to create gene maps of
different mammal species. Some species (such as mouse) are
inexpensive to keep in colonies and easy to breed, essential
factors for constructing a linkage map. For some, there is the
added benefit of available interspecies crosses to maximize
polymorphisms. Other species (such as lions and whales)
would not be so amenable to linkage analysis. Somatic cell
genetics has been particularly useful for mapping genes to
human chromosomes because of the characteristics of rodent-human hybrids, which rapidly segregate human chromosomes. Primate genes have been mapped by the same
strategy, as well as bovine and cat genes and genes on the
marsupial X. However, rodent genes are a challenge because
most hybrids perversely retain a full set of rodent chromosomes. Likewise, in situ hybridization has been most useful
for species that have easily distinguished chromosomes (such
as human, hamster, and various marsupial species) and for
which many cloned genes are available.
Progress in Constructing Linkage Maps
At the time of this writing, very detailed linkage maps
(with an average distance between markers of less than
0.01 cM) are constructed for mouse, with about 6000 genes
and 13,000 DNA markers mapped, largely through the
availability of interspecies crosses and backcrosses
(Davisson and others 1998). A linkage map for the rat has
been started more recently but now contains 900 known
genes and about 4000 microsatellites (Levan and others
1998). Major, mutually supportive efforts to construct good
linkage maps for livestock species have resulted in the
rapid expansion of maps of cattle, sheep, and pig. A medium density linkage map (more than 1400 markers) has
been constructed of the bovine genome (Womack 1998). A
map of the sheep genome with 519 markers (mostly
microsatellites) has been made; a 4.2-cM third generation
map of the sheep genome is under construction (Broad and
others 1998). The horse map is progressing rapidly, preliminarily with 150 markers (Bailey and Binns 1998). The
dog map has been started with more than 100 microsatellite
markers, but so far few coding genes (Binns and others
1998), and a 5-cM cat map has been derived from
interspecies crosses (O'Brien and others 1988).
Linkage mapping in other species has begun. A 10-cM
map of the baboon has recently been completed with 330
markers (Rogers and VandeBerg 1998). A framework linkage map is now available for the marsupial Monodelphis
domestica with 69 loci, half of them anonymous DNA markers (Samollow and Graves 1998).
54
Three chicken linkage maps at average densities of 6 to 9
cM containing up to 643 markers and 80 phenotypes have
been rapidly produced using reference crosses from Europe
and the United States (Burt and Cheng 1998). The growth of
linkage maps for different fish species has been even more
remarkable due to the special characteristics in some species
(for example, haploid progeny in zebrafish make backcrosses
unnecessary). Approximately 650 markers have been identified on the zebrafish map, including 100 coding genes
(Postlethwaite and others 1998), 334 markers (103 genes) on
the Xiphophorous map and 170 markers on the medaka map
(Morizot and others 1998). The beginnings of linkage maps
have been sketched out for several commercial species including trout and salmon.
The total size of the human genome is approximately
3700 cM, and that of other mammals is comparable. For
example, the mouse map is 1400 cM, the dog map is 2100
cM, and the sheep linkage map is 3500 cM. The chicken map
is approximately 3800 cM, although the genome is physically only about 1/3 the length of the mammalian genome.
The variation is more likely to reflect differences in rates of
recombination rather than large discrepancies in genome size.
Progress in Physical Mapping
Somatic cell genetics revolutionized human gene mapping,
establishing a framework for autosomal gene maps for the
first time. It also enabled gene mapping in great ape and
other primate species, for which no suitable breeding colonies existed. It provided the first autosomal maps for many
other species (such as bovine and cat; O'Brien and others
1998; Womack 1998) and is still used to build up the outline
of a physical map, even when higher resolution maps are
available (such as sheep; Broad and others 1998). More recently, somatic cell genetic analysis has provided a rapid and
relatively low cost means to assign largely type I markers to
chromosomes as a start to establishing horse and dog linkage
maps (Bailey and Binns 1998; Binns and others 1998).
Somatic cell genetic mapping is still the method of choice
for rapidly (and relatively cheaply) establishing a framework
map for species like the shrew or the vole (Nesterova and
others 1998; Serov and others 1998), in which no polymorphisms are available and breeding has been difficult. The
method has also been critical for building up maps for mink
(all 77 genes), fox (all 35 genes), and marsupials (the first 20
genes in the tammar wallaby) (Rubstov 1998; Samollow and
Graves, 1998; Serov 1998). However, the unstable karyotype and the chromosome fragmentation in cell hybrids produced between rodents and marsupials, monotremes and
birds has rendered the method less than ideal for mapping in
these groups, although other species combinations (such as
vole x M. domestica) appear more tractable for these distantly related species (Nesterova and others 1997).
Radiation hybrid mapping also has the advantage of requiring no intraspecific variation or breeding populations,
with the potential to order type I or II markers within small
ILAR Journal
regions. Radiation hybrid panels are now available for human, baboon (Rogers and VandeBerg 1998), bovine, pig,
mouse (Davisson and others 1998), chicken (Burt and Cheng
1998), and zebrafish and are under way for many other
species.
In situ hybridization is used in a growing number of species and is most useful where more accurate linkage or molecular mapping methods are unavailable. It requires the
availability of DNA clones, so is useful only in species in
which DNA libraries are available and at least a few genes
have been cloned. It is possible to use the low resolution, but
very sensitive, radioactive in situ hybridization to localize
heterologous clones, even from species as distant as human
and marsupials, monotremes, or chicken, as long as very
conserved genes are used (such as the human Duchenne
muscular dystrophy probe). Most of the gene assignments in
monotremes have relied on radioactive in situ hybridization
using human cDNA probes to conserved genes (Graves
1998).
Advances in fluorescence in situ hybridization have resulted in the greater sensitivity of this technique. FISH offers
better efficiency and resolution but requires long and almost
100% homologous DNA probes. However, in the great apes,
FISH with large insert human probes (cosmids and YACs)
has proved to be very effective. FISH has played a critical
role in anchoring unknown synteny or linkage groups to a
particular chromosome in many species. For example, 85
bovine linkage groups were located on 26 chromosomes by
FISH mapping 1 or more representatives, and the last of the
unknown bovine synteny groups finally found a home when
1 of the genes was located by FISH. With improved methods
for identifying chromosomes that are morphologically very
similar, more than 100 FISH localizations now exist in cattle
(Womack 1998), and the method has become useful even for
sheep.
Molecular mapping is far advanced in some species and
quite impracticable in others. Genomic and cDNA libraries
in phage, plasmid, and cosmid vectors have been constructed
from model mammals and livestock species, and even from
more exotic mammals like wallaby and platypus. Chromosome-specific libraries, constructed from DNA of sorted
chromosomes, are being developed for cattle (Womack
1998). Gridded YAC and BAC libraries are available for
cattle, pig, and sheep (Broad and others 1998) and BAC
libraries, for dog (Binns and others 1998).
Conserved Synteny in Mammalian Gene
Maps
Early comparisons at the cytogenetic level painted a nihilistic picture of complete genome scrambling between mammal groups. However, even the first comparative gene mapping—particularly between cat, bovine, and human—showed
a level of conservation that could not be appreciated using Gband comparisons (reviewed in O'Brien and others 1988).
The ensuing 2 decades of intensive mapping of hundreds of
Volume 39, Numbers 2 and 3
1998
loci across more than 40 species has consolidated a picture of
quite extraordinary genome conservation.
A giant jigsaw of more than 900 genes mapped in 32
species, constructed both from the data available in this issue
and in databases listed by Wakefield (1998), is presented as
the poster entitled "Comparative Genome Maps of Vertebrates" and enclosed in this issue (Wakefield and Graves
1998). Very large regions of conserved synteny with the
human genome are apparent when genes on the same chromosome are joined by vertical lines. For instance, most of
human chromosome 9 is represented by cat D4 and pig 15,
and human chromosome 17 is represented by mink 5, pig 12,
and bovine 14. Of the 23 human chromosomes, 16 are represented by a single cat chromosome, and the other 7 are split
between 2 cat chromosomes, representing a total of 30 homology segments if internal rearrangements are ignored
(O'Brien and others 1997). It is evident from this comparative map that large autosomal regions have been conserved
between human and primates, carnivores, artiodactyls, rodents, and insectivores.
Of all chromosomes, the X stands out as almost completely conserved between different eutherian mammals. Of
the hundreds of genes on the human X that have been mapped
in 1 or more of 37 other eutherian species, all are on the X
except 3 exceptional genes that map to the X in human and
an autosome in mouse. The exceptional conservation of the
X was recognized decades ago and was proposed to be the
result of selection against disruption of the chromosomewide X inactivation system (Ohno 1967).
Some exceptions to the picture of conservation of synteny
appear inconsistent with the evolutionary distance between
species. For instance, within the primates, which are generally very conserved compared with the human genome, the
gibbon stands out as having multiple breaks in synteny. In
addition, even with the few dog genes that can be compared,
it is evident that the dog map is more fragmented compared
with human than is the highly conserved cat genome.
Rodent maps are much more broken up with respect to
human maps. Even the earliest maps of mutants and isozyme
loci indicated that the conserved segments between the
mouse map and the human map are small. Detailed comparisons and subsequent analysis led to the conclusion that there
have been 150 rearrangements between the species, leaving
the average length of conserved segments as only 8.1 cM
(Nadeau and Taylor 1984). This limited conservation is even
more obvious now that the maps of both species are so detailed and is apparent from the comparative mapping poster,
even though not all of the mouse loci are represented
(Wakefield and Graves 1998).
It was initially thought that this breakdown of synteny
merely reflected the increased evolutionary distance between
rodents and primates. However, the hamster genome shows
considerably more synteny with human than does the mouse,
even though caviomorphs probably diverged from the primate lineage even earlier than rodents, and the same appears
to be the case for the even more distantly related shrew (Serov
and others 1998) and even chicken (Burt and Cheng 1998).
55
For instance, human chromosome 12 is broken up in mouse
and rat but is intact in the chicken genome, suggesting that
the mouse has a particularly rearranged karyotype that is not
typical of other orders and perhaps not even of other rodents.
Conserved Synteny in Distantly Related
Mammals and Other Vertebrates
Although marsupials and monotremes have genome sizes in
the range of eutherians, their karyotypes are very distinctive,
and it was initially expected that their genome arrangments
would be scrambled beyond recognition, by comparison with
the human genome. However, this is not the case. For example, 7 genes spanning human chromosome 17 all map to
linkage group 3 in M. domestica, and 7 human chromosome
3p genes lie on chromosome 2q in the wallaby (Samollow
and Graves 1998). Even in monotremes, the mammals most
distantly related to humans, conserved synteny is observed,
particularly in the X chromosome, which shows the same
distribution of most X-linked and autosomal locations
(Graves 1998).
Remarkably, conserved synteny is apparent, at least in
short regions, even in much more distantly related vertebrates. Birds, which evolved from a branch of reptiles that
diverged from mammals 350 million yr ago (MYA1), show
many shared syntenies of coding genes. For example, 8 human chromosome 6 genes all map to chick chromosome 3
(Burt and Cheng 1998). Even more remarkable is the conservation of synteny apparent in comparisons between humans
and fishes; for example, 10 markers on human chromosome
2q all lie within zebrafish linkage group 9 (Postlethwaite and
others 1998). Conservation of gene arrangement is all the
more remarkable in fish that have genomes only a fraction of
the size of the human genome. They contain the same genes,
having the same structure and similar sequence, and may
even be in the same order, although interrupted by much less
repetitive DNA. Vertebrates such as birds (with a genome 1/3
the size of mammal genomes) and especially pufferfish (with
a genome only 1/10 that of humans) provide an opportunity
to identify and sequence genes with much less labor.
Conserved synteny can give us an estimate of the number
of breakpoints needed to transform 1 map into another. This
is a minimum estimate, since internal rearrangements, such
as those described in the pig, will be detected only when
gene order is known. An even more dramatic representation
of such conserved synteny can be obtained directly by chromosome painting, which can also detect interruptions of
synteny within a chromosome.
Gaps in the Maps
The numerous gaps in the comparative map are very obvious. Some valuable genomes are poorly represented and include, surprisingly, nonhuman primates and the commercially important horse and dog.
56
Relatively few primate species have been analyzed thoroughly, despite early interest in primate gene mapping by
somatic cell genetics. There has been little ongoing interest
in somatic cell genetic mapping, and the suite of loci mapped
in different species is idiosyncratic and difficult to compare.
One difficulty has been the lack of accurate family data to
conduct linkage analysis, a problem now being corrected
with systematic studies of multigenerational pedigrees in
baboons (Rogers and VandeBerg 1998).
The paucity of comparative data for important domestic
species like horse and dog may be surprising but applies
more to the choice of markers than the absence of a map. Up
to the time of this writing, the emphasis in both species has
been on type I markers, and relatively few coding genes are
located whose homologues are obvious in other species. Inclusion of more coding genes will enable the rapidly developing linkage maps in these species to be aligned to the
human and other maps, to the mutual benefit of all species.
Not so surprisingly, more exotic species like shrew and
vole are poorly represented (presumably because we do not
eat them and because they are not commercially important)
and unfortunately the edentates—thought to have diverged
earliest in the eutherian radiation, are completely missing
(won't someone construct an armadillo or a sloth gene map?).
Farther afield, there have been sustained efforts to construct
good maps of commercial bird and fish species, but hardly
any data have been reported about reptiles, except for a few
assignments in the alligator.
Comparative Painting
Chromosome painting patterns are very much easier to compare between species than are the G-band homologies that
were the subject of enormous effort in the 1980s (Rofe and
Hayman 1985; Yunis and Prakash 1982). Although detailed
G-banding comparisons can potentially provide more information than comparative painting (such as about orientation
within conserved segments), in reality, 1 band looks much
like another in isolation, and it is possible to detect conserved banding patterns only in relatively large regions. Classic banding studies have therefore been useful in comparisons of closely related, but not distantly related, mammals.
The results from comparative painting between humans
and other mammals have been assembled for the first time
and are presented, with reference back to the human genome,
as the poster in this issue entitled "Comparative Chromosome Painting" (Glas and others 1998). Very large blocks of
conserved synteny are obvious throughout. Comparative
chromosome painting has largely confirmed the conclusions
of comparative mapping—that the genome has been very
conserved, at least between eutherian orders that diverged
about 60 MYA.
The extent of conservation within orders of eutherian
mammals is striking. For instance, 20 primate species have
been comparatively painted (Wienberg and Stanyon 1998).
Among the great apes, only 1 major rearrangement exists—
ILAR Journal
a fusion between 2 acrocentric chromosomes with homology
to human chromosome 2. The fused region has been pinpointed by using probes specific to regions of human chromosome 2. Some of the lesser apes, most notably the gibbon,
however, show multiple rearrangements (up to 40) compared
with humans.
Particularly striking are whole chromosomes that have
survived intact, not only within primates but also in other
orders. For example, of the 23 human chromosomes, 16 are
represented by a single cat chromosome and the remaining 7
by 2, mostly in uninterupted blocks (O'Brien and others
1997), confirming and extending the results from the comparative gene map. Conservation of entire chromosomes may
cover other orders; for instance, human chromosomes 4 and
17 appear intact in carnivores (cat and seal), artiodactyls (pig
and cattle), cetaceans (dolphin), and even insectivores
(shrew). The X chromosome is entirely conserved within
eutherians, as predicted by comparative mapping, although
autosomal additions form XY,Y2 systems in some species.
The minimum numbers of autosomal rearrangements
between human and other species can be assessed readily
from comparative painting patterns. A comparison of the
numbers of conserved autosomal blocks between human and
other species reveals 23 between human and chimp (that is,
only 1 rearrangment within the 22 human autosomes) The
human and cat genome share 32 conserved blocks (that is, 10
rearrangements) including 3 internal rearrangements, and
other carnivores about the same number (mink 34 and harbor
seal 31). Artiodactyls show somewhat more; for instance
bovine and human genomes share 56 autosomal blocks (that
is, 34 rearrangements). Remarkably, the common shrew, an
insectivore and probably as distantly related to primates as
any eutherian mammal, shares only 33 conserved blocks.
Painting between rodent species such as mouse and rat
reveals strong homologies and a minimum of 33 conserved
segments, implying at least 13 rearrangements (FergusonSmith and others 1998). However, relationships between the
rodent and the human genome are more complex, and definitive comparisons are not yet available, although FergusonSmith and others (1998) report that comparative painting
reveals 116 conserved blocks shared between mouse and
human.
Preliminary evidence from recent cross-species painting
in marsupials supports the hypothesis that the marsupial genome is even more conserved than the eutherian genome.
Marsupials have a few very large chromosomes, and painting has dramatically confirmed their close relationships
across species (Toder and others 1998).
PRACTICAL VALUE OF COMPARATIVE
GENOMICS
Since the mammalian genome is very conserved, it makes
sense to combine the gene mapping information from humans and nonhuman mammals for the mutual benefit of both.
Because excellent resources are available to the Human GeVolume 39, Numbers 2 and 3
1998
nome Project, both in funding and in the great variety of
phenotypes studied worldwide, the detail and quality of information about the human genome is unrivaled, probably
forever. Applying our knowledge of the human genome to
the genomes of other mammals, birds, and fish of interest as
sources of food, clothing, transport, or companionship, or as
valued environmental resources, will be of immediate value
in improving and conserving these resources. The availability of a detailed human map helps to establish an outline map
of any other mammalian species to which details may be
added and from which comparative information may be gathered. The resulting information is extremely valuable for
animal health and animal breeding.
Genetic information can flow in the opposite direction
equally well. Mapping information in a nonhuman species
can be directly transferred to the human gene map by reference to conserved chromosome regions. Many studies impossible in humans can be set up in model mammals, which
have many advantages for genetic study—including the possibility of genetic manipulation.
Applications to Animal Health
Information from the Human Genome Project can be applied
directly to veterinary research, diagnosis, and treatment. The
location and identification of disease genes in humans can
immediately assist in identification of the homologous condition in animals. If a similar phenotype maps within a
syntenic segment conserved between humans and a domestic
animal species, it is likely that a mutation in the same gene
causes the condition. This knowledge can be applied directly
to diagnosing the condition in animals. If treatment regimes
are already available for the homologous disease in humans,
they may be readily transferred to veterinary use. If the human gene has already been cloned, it is easy to clone the
homologous gene from any other animal, and to screen it for
mutations. Ultimately, the biochemical cause of the disease
may be understood and new treatment or a cure found.
Inherited diseases in dogs and horses are of particular
concern. Many dog breeds were developed from very small
founder populations subject to selection for different attributes of appearance or behavior and may be highly inbred.
Inherited eye diseases are particularly common. Some breeds
of horses have been selected over 100 yr for performance on
the racetrack, without much regard to health status. A number of diseases (such as bone diseases) are extremely common (Bailey and Binns 1998). Many diseases that are well
studied in humans (such as, anemias, muscular dystrophies,
immune deficiencies, cancer, and heart disease) occur in domestic animal species, and the knowledge we have gained of
the human conditions could have important benefits in animal husbandry.
One good example of the interaction of genetic studies in
human and livestock species is the identification and cloning
of the gene that controls malignant hypothermia, a condition
economically important in pigs and medically important in
57
humans. The discovery of a genetic factor that affected anesthetic response (malignant hyperthermia) in humans was followed by its identification as an economically important
stress syndrome in pigs and its location on the pig genetic
map. The conserved syntenic region in humans was searched
for candidate genes, resulting in the cloning of the human
ryanodine receptor gene RYR1 and subsequently, of the pig
homologue (Fujii and others 1991).
In addition to providing a direct comparison with human
diseases, comparative maps make possible the comparison
of disease states between livestock species or between livestock species and model mammals like the mouse (with, for
example, kidney diseases).
Animal Breeding
A detailed gene map of an animal species can assist animal
breeding, either indirectly by marker-assisted selection or
directly by cloning the genes responsible for phenotypes of
interest or concern—genetic diseases that are a problem or
economic traits that we seek to improve. Crosses are first set
up to establish genetic linkage of the phenotype to polymorphic markers on the map. Placement on the bovine map has
included several diseases as well as the polled gene, which
controls horn development (Womack 1998). Also mapped is
the potentially useful callipyge gene, which produces muscular hypertrophy in sheep hindquarters, and 2 genes that
affect fecundity (Broad and others 1998). When linkage is
discovered, closely linked markers can be used to predict the
phenotype of offspring, since the allelic combinations will
tend to be parental. Using marker-assisted selection, superior
animals may be chosen for raising and breeding at an early
stage (even before birth), and animals that will develop a
disease may be excluded from breeding.
Although marker-assisted selection has been of some
value, cloning the gene that confers the trait of interest is the
ultimate goal. A gene map may be used to isolate an unknown gene physically by a technique called positional cloning. One constructs a detailed linkage map and locates the
phenotype (desirable or undesirable) with respect to flanking
DNA markers by making crosses between animals that differ
in this trait. It is then necessary to switch reference to a
physical map, constructed by ordering cloned sequences of
DNA, to determine the position of the DNA markers flanking the disease gene on a physical piece of DNA (usually a
large insert clone like a YAC or cosmid, or a contig of overlapping pieces). The piece(s) of DNA containing the flanking markers must also contain the unknown gene, and they
can then be physically searched for sequences with the hallmarks of genes, or sequences transcribed into RNA in the
tissue of interest. Candidate genes may be identified and
sequenced in normal and variant animals to identify the one
revealing base alterations that correlate to the mutant phenotype.
The positional cloning procedure may be short circuited
by using a comparative candidate positional cloning ap58
proach. This shorter procedure entails locating the trait on a
linkage map in the species of interest and then scanning the
map of the syntenic region in a species with a high density
map (human or mouse) for genes that could be involved. For
instance, the fibroblast growth factor gene FGF1 was spotted in a region of the human map syntenic to a region of the
sheep map containing DNA markers linked to wool fiber
diameter differences. FGF1 was subsequently identified as
a major determinant of this important economic trait (Broad
and others 1998).
When a gene responsible for a disease or a superior trait
has been isolated, its base sequence is determined and translated (on paper or computer) into the amino acid sequence of
its protein product. The amino acid sequence will give us
some idea of the normal function of the gene, and the changes
in the variant animal will indicate what has gone wrong (or
right) in the mutant. Animal breeders will thus have tools to
diagnose a variant and to select for or against it. One example is the bovine leukocyte adhesion deficiency gene,
which was first mapped and then positionally cloned and
identified by mutation analysis (Womack 1998), enabling a
direct and easy PCR diagnosis of this economically important trait. Other examples, including the callipyge gene
(which may significantly improve meat yield in sheep) and
the polled gene (which may be used to select for or against
animals with horns), are now the subjects of intense searches
in cattle and sheep. Understanding the biochemical action of
a cloned gene offers the possibilities of additional improvement by selection or genetic manipulation.
Of particular significance for breeding domestic animals
is the capacity to locate, and subsequently isolate, genes that
have effects on economically important traits (economic trait
loci) like weight, meat quality, wool fiber, fertility, and fecundity. It was traditionally expected that practically all economically important traits are affected by many genes, each
with a relatively small effect (so-called quantitative trait loci,
or QTLs1). Identifying QTLs has been virtually impossible
by traditional breeding techniques because the differences in
phenotypes may be slight and often obliterated by environmental effects. An entire branch of genetic analysis (quantitative genetics) has developed to handle such data and use
the information in breeding practice. It is now evident that
QTLs can be mapped and genes with major effects can ultimately be identified and cloned just like any other gene. For
instance, linkage has been established for genes with major
effects on growth rate and fatness in pigs (Anderssen and
others 1994), as well as several QTLs for milk production in
the cow (Womack 1998). Information may be transferred
from 1 species to another, such as instances in which QTLs
affecting milk yield have been identified in syntenic regions
in sheep and cattle.
In addition to QTLs that confer an economic advantage,
a number of abnormal characteristics are determined by
QTLs and have been identified in dogs to include heart disease, dislocated hip, narcolepsy, and atopy. These can be
mapped in dogs and then the syntenic regions of the human
and mouse genome can be scanned for candidate genes.
ILAR Journal
Practical Benefits to Studies of Human
Disease and Development
Genetic information from domestic mammals, model mammals, or even other vertebrates can be of great value for
studies of the human genome and genetic disease. Information on maps or markers, and on genes involved with diseases or phenotypes of interest, may be available in a nonhuman species, and can be transferred directly to the human
gene map by reference to their position on conserved chromosome regions. Many studies impossible in humans can be
set up in model mammals. Nonhuman mammals present
many advantages for genetic study, including shorter generation time, large family size, and above all the ability to set up
crosses deliberately to study the transmission of genes over 3
or more generations. To extend these advantages, it is now
possible to genetically manipulate model mammals by targeted disruption of a gene ("knockout") or insertion of a
foreign gene (transgenesis).
Many disease states or traits with homologues in humans
are well studied in domestic mammals. More than 350 inherited recessive disorders are known in different dog breeds
that are particularly accessible to study because of the availability of large multigeneration pedigrees and because within
a breed, a particular mutant allele is certain to be identical by
descent (Binns and others 1998). For example, eye diseases
common in several dog breeds have human homologues that
can be studied much more easily in a dog model. Similarly,
sheep may provide models for cystic fibrosis, hemophilia,
Batten-Mayou disease, congenital cataract, and inherited
deafness (Broad and others 1998). It is sometimes advantageous to study a model for a human genetic disease in an
animal of a similar size and with similar physiology such as
sheep or pig. Many human diseases have homologues in 1 or
more animal species, and a detailed compendium (Mendelian Inheritance in Animals) has been developed to assist this
comparison (Nicholas and Harper 1996).
It has been important to develop disease models in rodents. More than 1000 spontaneous mutants in mice cause
diseases, and 100 of these have been proposed to be homologues of known human genetic diseases (Davisson and others 1998). For example, mutants responsible for inherited
deafness have homologues in mutants in several mouse
genes, as do several classic genetic defects such as Waardenburg syndrome and Hirschsprung's disease (CGOW 1996).
It is even possible to manipulate the genome in model
mammals to create mutants in a candidate gene and study
their effects on phenotype. Recessive diseases can be studied
by removing a candidate gene in mouse, either by selection
for a mutant embryonal stem cell line (such as producing an
//Pfl7-deficient "Lesch Nyhans" mouse) or the targeted disruption of the homologue of a human gene (such as the CFTR
gene to create a cystic fibrosis homologue). Dominant mutant alleles can be studied by introducing the mutant into a
mouse; for example, a Huntington's disease model was constructed by transfecting mice with a mutant human HD allele
having an expanded triplet repeat (Bates and others, 1997). It
Volume 39, Numbers 2 and 3
1998
is noteworthy that the knockout mutant does not necessarily
show a phenotype identical to that of the human disease
state.
Studies in model mammals are particularly valuable for
identifying quantitative traits. QTLs are difficult to identify
in humans but can be identified by crosses involving model
mammals or livestock. Several genes involved in diabetes,
obesity, epilepsy, and cancer susceptibility were first identified by crosses in rodents and then the information was transferred to human. As another example, crosses between 2
inbred strains differing in their susceptibility to colon cancer
resulted in the identification and mapping of 5 colon tumor
susceptibility genes (Davisson and others 1998). This knowledge will assist the identification of candidate genes in
syntenic regions in human families with inherited colon cancers. Genes involved in fat distribution and metabolism have
been mapped on the pig genome (Anderssen and others
1994). Likewise, behaviors exhibited by particular breeds of
dog can be followed by cross-breeding and may result in
some insight into human behaviors (Binns and others 1998).
Nonmammalian vertebrates have special practical uses.
Some species of oviparous vertebrates are particularly suitable for analysis of development because it takes place in an
easily accessible egg, rather than in an implanted mass deep
inside a heavily fortified uterus. The chicken has been an
important model for decades, and with the transparent zebrafish egg and embryo, it is easy to identify and study developmental mutants just by observing the living embryo within
the first few days after fertilization. Mutagenesis screening
has identified thousands of recessive developmental mutants
that exhibit specific defects such as dorsoventral patterning
of the primary embryonic axes, brain, cardiovascular, and
organ development, and these may be recovered and studied
(Ingham 1997), including even those with severe phenotypes
that would not survive even early pregnancy in human or
mouse.
DEDUCING THE ANCESTRAL MAMMALIAN
GENOME
One of the most exciting uses for comparative data is the
investigation of genome evolution in vertebrates. Wittingly
or unwittingly, all comparative geneticists—whether they are
working on sheep improvement or developmental mutants in
zebrafish—are contributing to our knowledge about how the
vertebrate genome evolved. This knowledge has great significance for our understanding of ourselves and our animal
relatives and for providing many practical benefits that result
from a deeper understanding of normal and abnormal gene
structure, organization, and function in mammals, including
humans.
Comparing genomes across evolutionary intervals provides another dimension to the Human Genome Project. Extrapolated backwards, such comparisons allow us to deduce
the form of the genome of common ancestors: of primates,
carnivores—and their common ancestor 60 MYA; or of eu59
therians, marsupials—and their common therian ancestor 130
MYA; of birds and reptiles—and their common ancestor 300
MYA; and ultimately of fish, reptiles, and mammals—and
their common vertebrate ancestor 400 MYA. We can chart
genome rearrangements that have occurred to separate lineages, and even the deeper events such as the genome duplications that have occurred at least twice in vertebrate evolution.
Vertebrate Phylogeny
To interpret comparative genomic data, it is necessary to
refer to a framework of relationships and approximate divergence dates provided by fossil evidence (Colbert and Morales 1991). A greatly simplified representation of the relationships between major groups discussed here is presented
in Figure 1. Increasingly, molecular phylogenies are being
constructed from masses of DNA sequence data, although
discrepencies between different data sets are still the subject
of much debate.
The 3 major groups of extant mammals include 2
infraclasses—Eutheria (placental mammals), and Metatheria
(marsupials), diverged about 130 MYA—and the subclass
Theria, which contains those diverged from subclass Prototheria
(the egg-laying monotremes) about 170 MYA. More than
3750 species of placental mammals in 16 extant orders are
distributed widely throughout the world. These orders radiated
rapidly from an insectivore-like ancestor in the Cretaceous 60
to 80 MYA, so it is difficult to determine the sequence of their
divergence (Figure 1). There are about 250 species of marsupials in 16 families in 3 orders (although higher order taxonomy
is the subject of perennial dispute). Marsupials are concentrated in Australasia (16 families), with a significant presence
in South America (3 families) and 1 North American species.
Australian and South American marsupials diverged about 80
MYA. All monotremes are confined to Australasia. There are
only 3 species in 2 families, which diverged 30 to 70 MYA.
Fossil evidence, as well as their anatomy and physiology, has
traditionally placed them as a separate subclass of Mammalia,
which diverged independently from the therian (eutherianmarsupial) line of descent about 170 MYA. However, it is
possible that monotremes are more closely related to marsupials than eutherians, and this idea received some support from
mitochondrial DNA sequence (Janke and others 1995).
Mammals diverged from a branch of reptiles (synapsids),
leaving no other descendants. They are therefore equally distantly related to the other 2 major branches of reptiles,
diapsids (snakes, crocodiles, and the ancestors of birds) and
anapsids (turtles), all of which diverged independently 300
to 350 MYA. Reptiles in turn diverged from amphibians,
which evolved from a branch of the fish 350 to 400 MYA.
Establishing Ancestral Gene Arrangements
Based on the foregoing descriptions of comparative genomic
data, it is thus possible to make comparisons at vastly differ60
ent evolutionary levels. Comparing the human genome with
those of the great apes, or the mouse genome with rat, can
inform us of recent changes within a lineage. Comparing the
primate genome with that of carnivores or artiodactyls provides information about changes over approximately 60
MYA, and comparisons between any of these groups and
rodents informs us of changes occurring in the last 80 million
yr. We can double the evolutionary distance by comparing
eutherian mammals with marsupials and treble it by looking
at monotremes. Then we can double it again by making comparisons between mammals and birds, reptiles, or fish.
By lining up the syntenic associations between members
of a species group, it should be possible to identify similarities and deduce the karyotype of their common ancestor. The
data are now available to begin this task, at least for the
common ancestors of primates, carnivores, and ungulates,
and perhaps even for the ancestral eutherian.
Karyotype arrangements shared by 2 species either could
be ancient and retained by both (shared ancestral, or plesiomorphic) or could be the result of a recent change within that
particular lineage (shared derived, or synapomorphic). Deducing ancestral arrangements of conserved chromosome regions
depends on distinguishing ancestral and derived arrangements.
This must be done with reference to an outgroup, a species
from a more distantly related group than that to which the
study species belong. For instance, cat or pig would provide a
suitable outgroup for comparisons between different primates,
whereas monotremes or chicken might be a suitable outgroup
for comparisons between marsupials and eutherians.
For chromosomes that show rearrangements between
human and another species, it is usually possible to deduce
the point at which the rearrangement occurred by comparison with a third group. For example, human chromosome 2
is represented by 2 acrocentric chromosomes in chimpanzee
and gorilla. Which state is ancestral? Did the change represent a fusion in the human lineage or a fission in a common
chimpanzee-gorilla lineage? Clearly the question is important, not only for deducing how human chromosome 2 came
into being, but also in establishing the relationships among
the 3 great ape species. The answer is clear because all higher
primates have the 2 acrocentrics, which must therefore be the
ancestral condition. Indeed, close examination of the sequences around the region reveals a relic of an abandoned
centromere close to the fusion point (Wienberg and Stanyon
1998). This scenario is also consistent with other data, suggesting that humans are more closely related to chimpanzee
than either is to the gorilla.
A good example of the application of these principles at
a deeper evolutionary level is the investigation of mamma-
FIGURE 1 Relationships of the major groups of vertebrates. Fossil evidence reveals a very approximate time scale for the divergence of reptiles from fish and amphibians, the divergence of mammals from synapsid reptiles, the divergence of the 3 major mammal
groups, and the divergence of the eutherian orders. AMPH, amphibians; NW, New World; OW, Old World.
ILAR Journal
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lian sex chromosome evolution (Graves 1995). Comparative
mapping shows that X chromosomes of eutherians are almost invariant. However, human X-linked genes fall into 2
very distinct groups in marsupials: Genes on human Xq and
the pericentric region are on the X in all marsupials, but
markers distal on the short arm are autosomal. This could
mean either that the ancestral X was large like the eutherian
X and lost a portion to autosomes in marsupials or, conversely, that the ancestral X was small like the marsupial X
and gained autosomal regions in eutherians. Appeal to a third
group of mammals, the more distantly related monotremes,
favors the latter possibility, since human Xp markers map to
similar autosomal clusters in the platypus.
The Big Picture
How near are we to describing the ancestral eutherian, marsupial, and monotreme genomes? An ancestral mammalian
genome? An ancestral reptilian or even vertebrate genome?
From the results of comparative gene mapping and comparative painting, it is possible to trace large sections of the
genome that are the same in closely and even distantly related eutherians. An ancestral primate genome, using shared
synteny between human, Old World, and New World monkeys, could be reconstructed, allowing identification of the
lineage-specific rearrangements such as the multiple rearrangements in the gibbon genome (Wienberg and Stanyon
1998).
Curiously, however, more rearrangements separate different primates than separate humans from some nonprimate
mammals. For example, 17 rearrangements exist between
human and lemur, but only 7 between human and cat, not
counting internal rearrangements. This immediately tells us
that many of the changes occurred in the lemur lineage and
that cat and human more closely represent the ancestral genome. This comparison is the first step in reconstructing a
putative primate-carnivore ancestral genome (Rettenberger
and others 1995). Confirmation of this high degree of conservation between the 2 orders comes from comparisons between human and other carnivores; mink with 8 rearrangements and seal with 10.
These comparisons may be extended to other orders.
More rearrangements (from 14 to 22) were described between humans and ungulates, at first suggesting a more distant relationship. However, the finding of only 7 rearrangements between humans and dolphin, a member of the whale
family now thought to have diverged more recently from
ungulates, implies that many of the differences observed in
syntenic associations and painting patterns between human
and sheep, pig, cattle, muntjac, and horse genomes are
likely to be specific for the artiodactyl and/or perissodactyl
lineage.
Adding rodent genomes into the equation complicates
the analysis immediately since more than 90 rearrangements
separate human from mouse and rat. Does this reflect simply
an increased divergence date? Comparison with more dis62
tantly related groups (outgroups) suggests that many of these
rearrangements occurred in the rodent lineage. For example,
human chromosomes 2 1 + 3 appear as a unit in every eutherian group except rodents and also appear to be intact in
marsupials. Ten genes on human chromosome 2 are split
between 2 mouse chromosomes, but all lie in the same linkage group in zebrafish. In agreement with this conclusion,
chromosome painting between human and the common
shrew (an insectivore, thought to be the most distantly related eutherian) identified only 33 conserved blocks, implying that primate and insectivore genomes differ by only 10
rearrangements.
It should therefore be easiest to deduce the form of the
genome of a common eutherian ancestor by comparing common chromosome blocks between the most conserved of the
distantly related species: human, cat and/or seal, dolphin,
and shrew. Inspection of the comparative painting poster
(Glas and others 1998) points to several autosomes that appear intact in each of these species (as well as in some or all
of the ungulates): These include conserved regions represented by human chromosomes 3, 6, 9, 11, 13, 17, 18, and
20. In addition, a number of human autosome regions are
associated in all other species, suggesting that they were
ancestral but disrupted in the primate lineage. For example,
associations of human 3/21, 14/15, and 16/19 are present in
cat, bovine/pig, dolphin, and shrew. In this way, it should be
possible to build a picture of the genome of an ancestral
therian that lived 80 to 130 MY A.
Deducing an ancestral genome in the other 2 branches of
mammals has been less of a challenge. Marsupials have a
few large chromosomes that made possible some of the most
thorough classical studies of karyotype evolution in any
mammal group. Extraordinary karyotypic conservation has
enabled an ancestral marsupial karyotype to be deduced by
cytological criteria alone, even before the discovery of
G-banding, certainly before comparative gene mapping had
much impact, and long before the advent of chromosome
painting. A "basic" 2n=14 karyotype, with nearly identical
G-band patterns, is represented within each of the major marsupial groups, and other marsupial karyotypes are easily
derived from it (Rofe and Hayman 1985). Even in the karyotypically diverse kangaroo family, karyotypes can be related
by Robertsonian fusions and fissions. The 2n= 14 basic karyotype itself may have been derived from a 2n=22 ancestral
marsupial karyotype (Svartman and Vianna-Morgante 1998).
Comparative gene mapping of marsupial autosomes is
not yet sufficiently advanced to test the hypothesis put forward by Rofe and Hayman, although at least the groupings
of autosomal genes appear to be the same in kangaroos and
dasyurids, which diverged about 45 MYA. Chromosome
paints have been prepared from marsupial species (FergusonSmith and others 1998) and painting is being adapted rapidly
to marsupial chromosomes. At the time of this writing, the
published results largely confirm (with some interesting exceptions) the predictions of Rofe and Hayman for kangaroo
karyotypes (Toder and others 1998).
Monotremes have a few large and many small chromoILAR Journal
somes, and the 3 extant species have karyotypes almost Gband identical (Graves 1998). The involvement of several
small unpaired chromosomes in a translocation chain at
meiosis (a feature unique among mammals) is also shared
among the 3 species, although the numbers of chromosomes
involved is different between the platypus and the 2 echidna
species. Comparative mapping has confirmed the same gene
arrangements on the X and 2 of the largest autosomes in
platypus and echidna.
Attempts to compare the maps of eutherians, marsupials,
and monotremes to deduce an ancestral mammalian karyotype are somewhat premature, given the paucity of autosomal markers on the maps. As yet it has not been possible to
paint autosomes across such vast evolutionary distances, although cross-species painting of the human X by the wallaby
X has been achieved (R. Glas, La Trobe University, Victoria,
Australia, personal communication, 1998).
Genome Duplication in Vertebrates
Can comparative gene mapping help us track the major
changes that occurred in the shaping of the vertebrate genome more than 400 MYA? It was suggested nearly 3 decades ago that genome duplication took place during the
early diversification of vertebrates (Ohno 1970). The confirmation since then of groups of paralogous genes in mammals
has been interpreted as evidence for 2 doublings of the vertebrate genome. However, it has been difficult to distinguish
polypoidization from tandem duplications of localized regions. Mapping 144 loci in the zebrafish has revealed very
large duplicated segments in both species, suggesting that 2
polyploidization events occurred before the divergence of
the mammal lineage from fish 450 MYA (Postlethwaite and
others 1998).
Evolution of Genetic Control Mechanisms
Genome Stability
The limited karyotypic change in marsupial evolution has
usually been regarded as an oddity of a weird group of mammals, and the conservation of the monotreme karyotype, an
artifact of the paucity of species. However, comparative mapping and painting now present a picture of an extremely
stable mammalian genome in which rapid change is the exception. Indeed, the conservation of synteny between human
and bird and fish maps suggests most strongly that the vertebrate genome is extremely stable. It is the variability of some
eutherian karyotypes—especially rodent—that is out of line.
Different eutherian groups show very different degrees
of genome stability. Primates show karyotypic similarities
identifiable by G-banding (as well as comparative mapping)
and beautifully confirmed by chromosome painting between
species that diverged as long as 50 MYA. However, the gibbon is exceptional, showing a dramatically scrambled gene
map as well as a rainbow of colored stripes on chromosome
painting (Wienberg and Stanyon 1998). Among the carnivores, the cat family has an almost invariant karyotype, but
the dog family reveals much variation. In the same way,
different marsupial groups evidence different levels of variation, from the dasyurids with almost no karyotypic variation
among many species, to the macropodids with a spread of
haploid numbers and chromosome morphologies. At the extreme are the rock wallabies, in which more than 20 different
karyotypes are found in a very rapidly diverging species complex (Toder and others 1998).
What is it that makes a karyotype stable? Is it in some
way an intrinsically good genome arrangement with some
sort of selective advantage (what?)? Or does something happen to destabilize the genome in 1 lineage? At the time of
this writing, we still do not understand the role of genome
change in speciation. Recent work suggests that interspecific
hybridization could play a role in rapid genome remodeling
by unleashing bursts of transposon activity (O'Neill and others 1998).
Volume 39, Numbers 2 and 3
1998
Comparative genomics can inform us not only about the evolution of genome arrangements but also about how different
genetic control mechanisms evolved and how they function.
Comparative studies of recombination, genomic imprinting,
X-chromosome inactivation, and sex determination provide
good examples of the advantages of comparative studies.
Linkage mapping depends on recombination, which occurs during meiosis in the male and female parent. Analysis
of the products of meiosis in the 2 sexes in humans and other
eutherians reveals a minor deficit in recombination in males.
However, this deficit does not appear to represent a general
rule of the influence of sex on recombination since in marsupials, females have far less recombination than males as the
result of a strongly sex-dependent distribution of chiasmata
(Bennett and others 1986). This major variation in chromosome behavior during meiosis may help clarify the molecular basis of initiation of recombination.
Knowing the physical location of imprinted genes and
studying them in more than 1 mammal species are critical to
the study of genomic imprinting. Genomic imprinting refers
to the expression of an allele according to its parental origin
(Tilghman 1992). For instance, the IGFII gene on the mouse
chromosome 7 is expressed only from the paternal copy (de
Chiara and others 1991); however, a nearby gene, H19, is
expressed only from its maternal copy. Intense mapping
around these loci in mouse has uncovered several other imprinted genes in the region, suggesting that parental imprinting is under regional control. The finding that these genes
also map together on human chromosome lip, and are also
imprinted, implies that imprinting evolved more than 70
MYA, and is therefore likely to be important for suvival or
reproduction. Several human genetic diseases are caused by
accidental derivation of both homologous chromosomes from
the same parent (uniparental disomy), mutation of imprinted
genes, or disruption of genomic imprinting. Mapping of the
same genes in marsupials and studying their expression will
enable conserved elements of the molecular mechanism (me63
thylation?) to be identified. These investigations will also
help us to determine when, and perhaps why, imprinting
evolved. Is it a dosage compensation mechanism? Or is it a
protection against parthenogenesis? Or does it reveal an
"arms race" between the male and female genomes to commandeer resources for the embryo?
X-chromosome inactivation is a large-scale control
mechanism affecting the activity of thousands of physically
linked genes on 1 X chromosome in females. Discovered in
1961, it is still unclear at the time of this writing how X
inactivation works. Mapping genes to the X in human and
mouse and studying their expression has been critical to
progress in understanding how this system of transcriptional
cis control—over megabases of DNA—is exerted by the
XIST locus on the X (Rastan 1994). Mapping the same genes
in other eutherian mammals, and particularly in marsupials,
has been important in understanding the molecular changes
that accompany inactivation, since marsupials show an incomplete, less stable inactivation and paternally imprinted
form that is probably ancestral (Cooper and others 1993).
Since there appear to be differences in the molecular mechanism between eutherian and marsupial X inactivation, comparisons will help to identify common elements (methylation? histone deacetylation?) common to the 2 systems. It
will also be critical in understanding why X inactivation
evolved: Was it simply a dosage compensation mechanism
to ensure fair play between XX females and XY males? Or
was it a primitive dose-dependent sex-determining mechanism?
Sex determination has been an area of intense study in
mammals, beginning with the search for the testis-determining
factor on the Y chromosome. Comparative gene mapping
and cloning, especially in human, mouse, and marsupial, has
played interacting roles in the search for testis-determining
factor and the investigation of how it, and other genes with
which it intracts, fulfills its role as trigger of the maledetermining pathway (Pask and Graves 1998).
CONCLUSION
Comparative genomics has advanced significantly since the
last comparative genomics report (Comparative Genomics
Organization Workshop 1996). At the time of this writing,
genetic and physical maps are being constructed for a variety
of species using a variety of methods, and comparative chromosome painting has been applied very rapidly to cytogenetic comparisons of homology.
Genetic mapping is well advanced in several livestock
species as well as rodent model species, and outline maps are
available for at least 30 mammal species in 8 orders. Maps
are being constructed rapidly for chicken and fish species.
Although the level of detail between maps of different species may differ by 2 orders of magnitude, it begins to be
possible to compare the genomes at a greater or lesser level
of resolution. Arrangements may then be compared across
mammal—and even other vertebrate—species.
64
Comparative mapping enables transfer of information
between human, livestock, and rodent genomes. Comparisons of location can be used to identify homologous genes
involved in disease states in humans, domestic animals, and
rodent models, making possible the development of techniques for diagnosis and treatment for transfer in either direction. Pinpointing a genetic trait on a linkage map is the
first step to physically isolating it; and when it is isolated
from 1 species, it may be obtained from any other by homologous cloning. This enables us to identify genes that
control genetic diseases, and even makes it realistic to consider cloning genes involved in quantitative traits (economic
traits like weight or milk yield, as well as diseases like cardiovascular disease or cancer). Comparative gene mapping
can therefore deliver information for the benefit of research
into animal health and animal breeding. It should greatly
speed up the search for genes that specify inherited diseases
in mammals and humans, as well as genes that specify economically important traits.
Comparisons, even over vast evolutionary time scales,
show that the mammal genome—indeed, the vertebrate genome—has been very conserved. Thus it now becomes possible to ask how the mammal genome—even the vertebrate
genome—evolved, and how it works.
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